The invention relates to a device and a method for producing, from any previously configured ion beams, precisely localized small packages of ions which all fly at the same velocity.
The use of mass spectrometric methods in biochemistry, particularly in genetic and protein research, is still limited by the fact that a large amount of substance is consumed when using these methods. Lower substance consumption is also demanded for other applications. In order to obtain a mass spectrometric reading from a few attomols of a substance (1 attomol=600,000 molecules), substance ionization must be maximized and ion losses must be reduced to a minimum during every stage from ion generation up to the actual measurement. The yield must be optimized at each step.
In this regard, a particularly crucial step is how the ions are injected into the mass spectrometer being used since with the different types of mass spectrometer, such as the ion-trap or time-of-flight mass spectrometer with orthogonal injection this still cannot be achieved with losses near to zero.
The production of ions for mass spectrometric analysis inside a vacuum system has the disadvantage of requiring a large excess of the substance molecules to be introduced into the vacuum system. On the one hand, there is the risk of contaminating the ion source due to the molecules of the substance condensing on the walls, thus giving the surfaces a charge and impairing operation. On the other hand, the ion yield from the ionizing processes inside the vacuum is very low. For this reason, ions are now being produced more and more outside the vacuum system of the mass spectrometer and then transferred to the mass spectrometer by using suitable methods.
Among the ion sources external to the vacuum which are available are, for example, Electrospray Ionization (ESI), in which substances with exceptionally high molecular weights can be ionized with very high yields. The electrospray ionization is frequently coupled with modern separation methods such as liquid chromatography or capillary electrophoresis. This group of ion sources external to the vacuum also includes methods using Inductively Coupled Plasma (ICP) ionization, which is used in inorganic analysis. Finally, there is Atmospheric Pressure Chemical Ionization (APCI) utilizing primary ionization of the reactant gases by corona discharge or beta emitter with electrons emitted at low energy. APCI is also used for the analysis of air pollutants and is particularly suitable for coupling to mass spectrometry via gas chromatography, liquid chromatography and capillary electrophoresis. Other types of ion sources external to the vacuum, such as Grimm hollow cathode discharge or matrix assisted desorption to air, are still being examined and developed.
The practice so far has been to release the ions from these sources along with large quantities of environmental gas into the vacuum of the ion-trap mass spectrometer. Fine apertures of approx. 30 to 300 xcexcm diameter or 10 to 20 cm long capillaries of approx. 500 xcexcm internal diameter are used for this purpose. The excess gas must be removed by means of differential pump stages. Commercially available mass spectrometers use two or even three differential pump stages with the corresponding number of chambers upstream of the main chamber of the mass spectrometer. Three to four pumps are therefore used. The chambers are connected only by very small apertures and the ions are transported through these tiny apertures.
Where only two differential pump chambers are used in commercially available mass spectrometers, the pressure in the first differential chamber is usually a few millibar; in the second differential chamber the pressure falls to 10xe2x88x923 to 10xe2x88x921 millibar and does not drop to between 10xe2x88x926 and 10xe2x88x924 millibar until the main vacuum chamber. The mass spectrometer is located in the main vacuum chamber. The ions have to be transported through the differential pump chambers and through the tiny apertures between the chambers. During this process, there are considerable losses.
High-frequency multipole ion guides are often used to transport these ions through the chambers. The ion guides can only be used in the second differential pump chamber or in the main vacuum chamber because they are favorably used at a few 10xe2x88x923 millibar, as they then rapidly dampen both radial oscillations and longitudinal movements and thereby provide relatively favorable conditions for further ion transport and analysis in the mass spectrometer.
The U.S. Pat. No. 5,818,055 (DE 196 28 179, Franzen) describes ion packaging in an n-phase travel field where the phases are applied sequentially to annular electrodes which are equally spaced along and concentric to the axis. According to this patent: xe2x80x9cThe travelling field can be produced within a package of coaxially arranged annular discs. An n-phase rotational RF voltage must be generated for this purpose and the phases connected cyclically in series to the annular discs. For example, if a 6-phase alternating voltage is generated, the first phase will then be connected to annular discs 1, 7, 13 and 19 etc. and the second phase will be connected to annular discs 2, 8, 14 and 20 etc. In this way, an electrical travelling field is created in a known way within the package of annular discs, and potentials of the same phases move along the axis of the package. When a potential minimum is filled with ions at the start of the package of annular discs, then this potential minimum moves along the axis of the package carrying the ions along with it. With this arrangement, the ions are initially accelerated until a velocity equilibrium has been established. Here, the damping gas can help damp the oscillations of the ions around an average velocity.xe2x80x9d
Since then, it has been found that, in this system, precisely when the ion package has acquired the velocity of the travelling field, radial focusing for the package is no longer possible. In flight, the ions are always in phase with the electrically attractive diaphragms which they pass flying, and therefore are defocused continuously by the attractive forces of these apertures. Radial focusing will only take place when each particle experiences a surrounding radially retroactive pseudo-potential, as already described in U.S. Pat. No. 5,572,035 (Franzen).
The use of ideal axis-focused packages (when they can be produced) for injection into an RF quadrupole ion trap has already been described in the patent mentioned, DE 196 28 179 or U.S. Pat. No. 5,818,055. However, it would also be possible to use ion packages such as these for injection into the cells of an ion cyclotron resonance mass spectrometer (often simply referred to as a Fourier-Transform mass spectrometer). These types of ion packages can also be used to advantage for time-of-flight mass spectrometers with orthogonal ion injection.
Time-of-flight mass spectrometers with orthogonal injection of the primary ion beam have a so-called pulser at the beginning of the flight path which, according to the technology used so far, accelerates a section of a continuous primary ion beam (i.e. a thread-shaped ion package) at right angles to the previous beam direction into the time-of-flight mass spectrometer. A ribbon-shaped secondary ion beam is formed at the same time in which light ions travel fast and heavy ions travel more slowly. The direction of flight of this beam is located between the previous direction of the primary ion beam and the direction of acceleration oriented at right angles to it (see FIG. 4). This type of time-of-flight mass spectrometer is preferably run with a velocity-focusing reflector which reflects the entire width of the ribbon-shaped secondary beam and guides it to a detector which is similarly widened. Just such a mass spectrometer with a gridless optical system is described in Patent application DE 100 05 698.9 (Franzen).
The mass resolution of a time-of-flight mass spectrometer such as this essentially depends on the spatial and velocity distribution of the ions in the primary beam in the pulser. However, it also depends on the parallel adjustment of the pulser, reflector and detector since the slightest error in the parallel adjustment of the pulser, reflector or the detector results in operating time differences which are bound to lead to a reduction in the mass resolution. Apart from this, for sequential pulses, not all ions in the primary beam can be measured in the mass spectrometer since the pulser can only be filled according to either the heavier and slower or the lighter and faster ions.
A time-of-flight mass spectrometer with orthogonal ion injection is mainly operated with ion sources which produce large molecular ions from substances which are of biochemical interest. Ionisation is achieved by, for example, Matrix Assisted Laser Desorption and Ionization (MALDI) or by electron spraying of dissolved substances under atmospheric pressure outside the vacuum system (ESI=Electron Spray Ionization). In the latter case, the ions are introduced into the vacuum via input apertures or input capillaries and the accompanying gas (usually nitrogen) which is admitted with them is removed in several differential pump stages; see for example U.S. Pat. No. 6,011,259 (Whitehouse et al.).
Ions which are produced by MALDI, ESI or some other ionizer are injected into an ion guide system somewhere en route to the time-of-flight mass spectrometer, the principle of which is shown in FIG. 4. This can be carried out at an early stage in one of the differential pressure steps, in which case the ion guide system can pass through the walls between the differential pressure steps. However, this can also take place later in a special vacuum chamber, as shown in FIG. 4. During injection, the ions generally possess a certain kinetic energy of a few electron volts which they have mainly picked up from an electrical guide field and which is used to transport them into the ion guide system. The energy must not exceed approx. 2 to 8 electron volts if fragmentation of the ions by subsequent collision in the ion-guide system is to be avoided.
An RF ion-guide system is able to keep ions of moderate energy and not too small mass away from an imaginary cylinder wall of the ion-guide system (see also U.S. Pat. No. 5,572,035). The ions are injected, as it were, enclosed as in a pipe. This effect is achieved by using a so-called pseudo-potential field, a time-averaged force field which acts on the ions. (The pseudo-potential is mass dependent which, in this case, is only of marginal interest.) The pseudo-potential of all previously known ion guide systems has a trough at the axis of the ion guide system and increases towards the imaginary cylindrical wall. It reflects ions which approach the imaginary cylinder wall.
Time-of-flight mass spectrometers with orthogonal injection require the injected ion beam to be conditioned to an extremely high level. Here too, packaging the ions would be an advantage. Until now, the ion beams have been conditioned by using so-called ion guides which are filled with damping gas to dampen the axial movement of the ions. These gas-filled ion systems are also used for fragmenting selected xe2x80x9cparent ionsxe2x80x9d by collisions with the damping gas. The ionized fragments of the parent ions are called xe2x80x9cdaughter ionsxe2x80x9d.
External types of ionization such as electrospray often produce both singly charged ions and polycharged ions. Mass spectrometers only measure the so-called mass-to-charge ratio, i.e. the mass (usually expressed in atomic mass units) divided by the charge (usually expressed as the number of elementary charges). In the following, this mass-to-charge ratio will be referred to simply as the xe2x80x9cspecific mass.xe2x80x9d
The invention starts from a system of coaxial annular electrodes described in U.S. Pat. No. 5,572,035 where a series of annular electrodes are alternately connected to the two phases of an RF alternating voltage. When this system is filled with damping gas at a suitable pressure, axially injected ions are decelerated and then collected at the axis of the system. However, the system does not provide further propulsion for the ions. As described in the patent cited, such propulsion can be provided by, for example, a superimposed dc voltage to produce a fine, continuous ion beam of very small cross section.
In U.S. Pat. No. 5,818,055 (equivalent to DE 196 28 179), the idea was described to use an electrical travelling field on the system instead of the RF voltage, however, the ions are no longer focused in the axis as soon as the ions assume the speed of the travelling field.
To overcome this deficiency, in this invention two superimposed alternating voltages are applied to the annular electrodes: firstly an alternating two-phase RF voltage (for example, 40 V at 5 MHz) and secondly a multiphase low-frequency voltage (for example, 50 V at 50 kHz with six phases), forming a travelling field with an advancing rotational angle of the phases. The RF voltage provides the axial focusing and the low-frequency travelling field provides the packaging and transport of ion packets to the end of the system of annular diaphragms. Here, all ions of different specific masses are propelled at the same speed. In order to achieve superimposition with the two phases of the RF voltage, the number of rotational phases of the travelling-field voltage must be even. At least four, but preferably six, eight or more phases must be present. Preferredly, the phase angle between the phases should be equal. The condition of an even number of phases was not required in U.S. Pat. No. 5,818,055.
In an annular electrode system with aperture diameters of approximately six millimeters and where the electrodes are equally spaced at three millimeters, a six-phase travelling field at 50 kHz provides a travelling velocity for the ion packages of 900 meters per second. One ion package is ejected every 20 microseconds. The ion packages are spaced at 18 millimeters from each other.
At the axis of the apertured diaphragm system, the RF voltage is barely discernible. The low frequency travelling field voltage, on the other hand, is clearly present in the axis of the system although only with a fraction of the potential which the travelling field voltage produces at the diaphragms themselves. The potential wave depth drifting along the axis is dependent on the distance between the diaphragms and the aperture diameters of the annular diaphragms.
The injection of ions at low injection energy (to avoid fragmentation) into the more slowly moving waves of the travelling field voltage at the axis can be difficult. This can be helped by initially increasing the travelling field voltage at the start of the annular diaphragm system slowly by ramping the voltage amplitude maxima. However, this type of ramp is electronically difficult to create.
Nevertheless, an apertured diaphragm system can be made to have a similar effect to a voltage ramp. The spaces between the diaphragms in the apertured diaphragm system must be very small with a large aperture diameter at the beginning but, towards the end, the spaces must increase and the aperture diameters must decrease. The potential wave depth at the axis is then small at the beginning but increases towards the end. It is then easier to inject low-energy ions. Systems where only the spaces or the aperture diameters are varied are not so effective. An annular system in which the spaces between the annular diaphragms at the beginning are small but increase towards the end, accelerate the ion packages towards the end.
The ion packages can be injected, for example, into an RF quadrupole ion trap in phase, as described in DE 196 28 179. But they can also be injected into the pulser of a time-of-flight mass spectrometer where the time-of-flight mass spectrometer can be operated with an out-pulse frequency of, for example, 50 kHz. Here, because of the packaging, all the ions of an ion beam are used for the analysis. For this simple method of operation, the pulser need only be very short. However, the out-pulsed ions then spread out widely according to their specific masses since the deflection angles in the pulser are different for the different specific masses. A wide reflector and a much wider ion detector than those required for operation with a continuously injected, non-packaged ion beam are therefore still necessary.
However, a system can be set up which operates with a pulser of medium length, a relatively narrow reflector and a relatively short detector. The ion packages have to be subjected to lower post-acceleration so that the lightest ions will be faster than the heavier ions and will penetrate further into the pulser. However, the angle of deflection during out-pulsing is larger. It is therefore possible for the lighter ions to hit the detector at the same point as the heavy ions in the package. However, it is not possible to focus ions of all specific masses on one point of the detector, although the required length of the detector will be significantly smaller than for conventional operation.
The main advantages of these methods are as follows:
since all ions in the ion beam are used, the sensitivity is high;
since the ions of one specific mass always start from the same small site in the pulser and always hit the detector at the same spot, the mass resolution is largely insensitive to small maladjustments of the parallelity of the pulser, reflector and detector.
The damping-gas filled apertured diaphragm system according to the invention with axial focussed RF voltage and propelling travelling field can, in particular, also be used for fragmenting selected parent ion species. The parent ions can be selected in a mass spectrometer, such as a quadrupole filter, connected upstream. They are then injected into the apertured diaphragm system with an energy (e.g. acceleration at approx. 30 to 50 volts) which is sufficient for them to collide with the molecules of the damping gas and fragment. The pressure of the damping gas is raised high enough for the ions in the gas to be slowed to a standstill in the absence of a travelling field. However, the travelling field takes over all the remaining parent ions and newly formed daughter ions and guides them to the end of the apertured diaphragm system.